The investigation of cell lines derived from human teratocarcinomas can provide information pertinent to human embryonic cells and to the clinical management of these tumors. Progress depends upon characterizing the properties of the stem cells (presumed to be the embryonal carcinoma (EC) cells) and the cells into which they can differentiate. We have succeeded in cloning two human EC cell lines. One of these, 2102Ep, differentiates to a limited extent in culture, probably along a trophoblastic lineage. From studies of this line, confirmed by immunohistochemical examination of tumor biopsies, we have shown that human EC cells express the cell surface antigen SSEA-3, but not SSEA-1, contrasting with murine EC cells which are SSEA-3-/SSEA-1+. Further, HLA-A,B,C and beta-2-microglobin are expressed at low levels by human EC cells, which also synthesize little or no fibronectin. Agents such as retinoic acid have no detectable effect on these cells, but differentiation may be induced by low cell density, when SSEA-1 becomes expressed, SSEA-3 is diminished and fibronectin synthesis is induced. Cell sorting experiments indicate that it is the SSEA-1+ cells which synthesize the fibronectin. Other data also have shown that the fibronectin produced has a slightly higher molecular weight than fibroblast fibronectin and could represent an embryonic variant. A panel of monoclonal antibodies raised against these cells include antibodies reacting with antigens of restricted cell distribution as well as others expressed by most human cells. These are currently being further characterized with the object of identifying additional markers that may be useful in distinguishing between human EC cells and their derivatives. The second human EC cell line that we have cloned is now being studied further to establish whether the characteristics identified for the 2102Ep system, which differ substantially from the characteristics of murine EC cells, may be generally applicable to human EC cells in culture. Our preliminary data show that this second line is capable of much more extensive differentiation than the other human teratocarcinoma cell lines that are available to us.